*/Disease Information (sort of like the Intro to your Mini Research Write up): Clostridium botulinum is the bacteria that causes botulism. Botulism is an intestinal disease caused by undercooked food, entry into wounds, or inhalation of spores. Botulism is highly preventable, as food-bourns botulism dies attemperatures above 185 degrees F and most wound botulism is the product of street drug injections. Botulism is generally treated with antibiotics, which have a moderate success rate, leaving about a 5-10% mortality rate. Most issues with botulism arise from misdiagnosing the symptoms as a common virus, allowing the bacteria to grow strong enough that it isn't eradicated by antibiotics, or simple lack of antibiotic availability (non-issue in first-world countries).
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): The best known of these is leupeptin (N- acetyl-L-leucyl-L-leucyl-D,L-argininaldehyde), which was originally isolated from Streptomyces exfoliates and inhibits a wide range of serine, cysteine and threonine-type peptidases, including trypsin, PACE4 calpain, clostripain and the trypsin-like activity of the proteasome (Aoyagi et al., 1969; Kembhavi et al., 1991; Kurinov and Harrison, 1996; Mains et al., 1997; Moldoveanu et al., 2004; Savory et al., 1993).
--- List cost and quantity of substrate reagents, supplier, and catalog # Clostripain (Endoproteinase-Arg-C); 10 ug; LS02135; $83.00.
Structure (PDB or Homology model) -- PDB # or closest PDB entry if using homology model: 4YEC
Current Inhibitors:
EDTA
Oxidizing agents
Sulfhydryl reagents (such as TLCK) (Porter et al. 1971)
Co2+, Cu2+, Cd2+, and heavy metal ions
Citrate, borate and Tris anions partially inhibit
Expression Information (has it been expressed in bacterial cells): Clostripain is expressed by the anaerobic bacteria Clostridium histoliticum. Clostripain has also been expressed and isolated from Escherichia coli and Bacillus subtilis.
Purification Method: cation-exchange chromatography and ultrafiltration
Image of protein (PyMol with features delineated and shown separately): *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): METPEPRTTRTILVYMMANNSLNSFASKNIESMIEGATSKNLNGGNLIVY EEE EEEEEEE SS TTHHHHHHHH HHHHHH HH HHTT EEEEE YAPAGSPPELLRIKEENGVVKKIHLKDYEK QNSADPDVMRSVIGEVVSQY EE SSS EE EEEEEETTEE EEEEEEE S TTSHHHHH HHHHHHHHHS PADSYGLVLWSHGTAWLPSDYQNKLK EEEEEEEEE S B TT TT GGGG
*length of your protein in Amino Acids 126
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website 53.0 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength: 87,890 cm-1 M-1
*GC% Content for gene: *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences): **
*NCBI Gene # or RefSeq#: 5397352
*Protein ID (NP or XP #) or Wolbachia#: 4YEC
*Organism (including strain): Clostridium botulinum
Etiologic Risk Group (see link below): 2
*/Disease Information (sort of like the Intro to your Mini Research Write up):
Clostridium botulinum is the bacteria that causes botulism. Botulism is an intestinal disease caused by undercooked food, entry into wounds, or inhalation of spores. Botulism is highly preventable, as food-bourns botulism dies at temperatures above 185 degrees F and most wound botulism is the product of street drug injections. Botulism is generally treated with antibiotics, which have a moderate success rate, leaving about a 5-10% mortality rate. Most issues with botulism arise from misdiagnosing the symptoms as a common virus, allowing the bacteria to grow strong enough that it isn't eradicated by antibiotics, or simple lack of antibiotic availability (non-issue in first-world countries).
Link to TDR Targets page (if present):
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.):
http://www.ncbi.nlm.nih.gov/pubmed?SUBMIT=y&term=3.4.22.8%5BEC%2FRN%20Number%5D
Essentiality of this protein: pathogenesis
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): multimer (di-mer)
Complex of proteins?: no
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
The best known of these is leupeptin (N- acetyl-L-leucyl-L-leucyl-D,L-argininaldehyde), which was originally isolated from Streptomyces exfoliates and inhibits a wide range of serine, cysteine and threonine-type peptidases, including trypsin, PACE4 calpain, clostripain and the trypsin-like activity of the proteasome (Aoyagi et al., 1969; Kembhavi et al., 1991; Kurinov and Harrison, 1996; Mains et al., 1997; Moldoveanu et al., 2004; Savory et al., 1993).
*EC#: 3.4.22.8
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/enzyme.php?ecno=3.4.22.8
-- Show screenshot of BRENDA enzyme mechanism schematic
*Enzyme schematic was not offered on BRENDA page
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
-- link to Sigma (or other company) page for assay (see Sigma links below) -- or link (or citation) to paper that contains assay information
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/clostripain.pdf
-- links to assay reagents (substrates) pages.
http://www.worthington-biochem.com/CP/assay.html
--- List cost and quantity of substrate reagents, supplier, and catalog #
Clostripain (Endoproteinase-Arg-C); 10 ug; LS02135; $83.00.
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 4YEC
Current Inhibitors:
Expression Information (has it been expressed in bacterial cells):
Clostripain is expressed by the anaerobic bacteria Clostridium histoliticum. Clostripain has also been expressed and isolated from Escherichia coli and Bacillus subtilis.
Purification Method:
cation-exchange chromatography and ultrafiltration
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
METPEPRTTRTILVYMMANNSLNSFASKNIESMIEGATSKNLNGGNLIVY
EEE EEEEEEE SS TTHHHHHHHH HHHHHH HH HHTT EEEEE
YAPAGSPPELLRIKEENGVVKKIHLKDYEK QNSADPDVMRSVIGEVVSQY
EE SSS EE EEEEEETTEE EEEEEEE S TTSHHHHH HHHHHHHHHS
PADSYGLVLWSHGTAWLPSDYQNKLK
EEEEEEEEE S B TT TT GGGG
*length of your protein in Amino Acids
126
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website
53.0 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength:
87,890 cm-1 M-1
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
GCTATAATCAATGCCATATATATAAAGATTAAAGTAATCTTTTGTATCATCTAATAATGG
AGTATCAGCTTTATTAATTAATTGCTTTTTTAAAAAATTATTTGTAAGTTAGTATACTCT
TCTACTAAATAGTTAAATTCAGCTTTTCATTTTTCCATCACTACCATAATTACGTAATCA
TTATATCATAGAATGCTTTTACCACGTAATATAAAAAGCAAGAGTCCTATATTAAATCCT
TGAACCTAAAGACCTAATGTAGATTTTTAACAAAACAAATGATTGATCACTTAATATATC
TAACAGTTCAGATAACTTCCTTTTTGAGATTGTAATATATAAGTAAATTCAAAGACCATA
AAATATACCCCCCGATTGTAATGCTTGAATATTAATTATAATGAAATTATAGTTTAAATT
AAAAAAATAATTTTTTTATGAAGTTTAAGAAGGATTATCCTAAACATTATAGAATATATT
AAATATAAAAATAATGTAAAACTTTAAATAATAACTCTTATAATGGTACATAATGTATTT
ATATGGATTAAATTTTAATTTATAGATGCTATAAAACTATAAATTAAATTTAAAATATAC
TTTTGGAGGTATGTATTATGTTAAGAAGAAAAGTATCAACACTATTAATGACAGCTTTGA
TAACTACTTCATTTTTAAATTCCAAACCCGTATATGCAAATCCAGTAACTAAATCCAAGG
ATAATAACTTAAAAGAAGTACAACAAGTTACAAGCAAGAGTAATAAAAACAAAAATCAAA
AAGTAACTATTATGTACTATTGCGACGCAGACAATAACTTGGAAGGAAGTCTATTAAATG
ATATCGAGGAAATGAAAACAGGATATAAGGATAGTCCTAATTTAAATTTAATTGCTCTTG
TAGACAGATCCCCAAGATATAGCAGTGACGAAAAAGTTTTAGGTGAAGATTTTAGTGATA
CACGTCTTTATAAGATTGAACACAATAAGGCAAATAGATT
*GC% Content for gene:
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**